Expression of active human factor ix in mammary tissue of transgenic animals

ABSTRACT

Recombinant Factor IX characterized by a high percentage of active protein can be obtained in the milk of transgenic animals that incorporate chimeric DNA molecules according to the present invention. Transgenic animals of the present invention are produced by introducing into developing embryos DNA that encodes Factor IX, such that the foreign DNA is stably incorporated in the DNA of germ line cells of the mature animal. Particularly efficient expression was accomplished using a chimeric construct comprising a mammary gland specific promoter, Factor IX cDNA that lacked the complete or any portion of the 5′-untranslated and 3′-untranslated region, which is substituted with a 5-′ and 3′-end of the mouse whey acidic protein gene. In vitro cell cultures of cells explanted from the transgenic mammal of the invention and methods of producing Factor IX from such said culture and methods of treating hemophilia B are also described.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of application Ser. No. 11/117,705filed on Apr. 29, 2005, now U.S. Pat. No. 7,419,948, which is acontinuation of U.S. patent application Ser. No. 10/062,447 filed onFeb. 5, 20002, now abandoned, which is a division of U.S. patentapplication Ser. No. 09/367,087 filed on Sep. 15, 1999, now U.S. Pat.No. 6,344,596, issued Feb. 5, 2005, which is a 371 of PCT/US98/02638,filed Feb. 13, 1998, which claims benefit of U.S. ProvisionalApplication No. 60/037,145 filed Feb. 14, 1997. This application claimsonly subject matter disclosed in the parent application and thereforepresents no new matter.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the production of natural and modifiedforms of Factor IX. In particular, the invention relates to a transgenicanimal containing, stably incorporated in its genomic DNA, an exogenousFactor IX gene that is expressed specifically in mammary tissue, suchthat Factor IX is secreted into milk produced by the animal. Inparticular, the invention relates to the production of human Factor IXin the milk of a transgenic non-human mammal using a DNA molecule thatcomprises a whey acidic protein promoter gene, 5′ regulatory sequencescontaining the promoter, human Factor IX cDNA that lacks at least aportion of the complete or any portion of or the complete the3′-untranslated region of the native human Factor IX gene, but containsthe 5′ and 3-′untranslated region of the mouse whey acidic protein.gene.

2. Background

Human Factor IX, or “Christmas factor,” is encoded by a single-copy generesiding on the X-chromosome at q27.1. For a review of Factor IX genestructure and expression, see High et al., “Factor IX,” in MOLECULARBASIS OF THROMBOSIS AND HEMOSTASIS, High (ed.), pages 215-237 (Dekker1995); Kurachi et al., Thromb. Haemost. 73:333 (1995). The Factor IXgene is at least 34 kilobase (kb) pairs in size, and it is composed ofeight exons. The major transcription start site of the Factor IX gene inhuman liver is located at about nucleotide −176. The human Factor IXmRNA is composed of 205 bases for the 5′ untranslated region, 1383 basesfor the prepro Factor IX, a stop codon and 1392 bases for the 3′untranslated region.

Factor IX is synthesized as a prepropolypetide chain composed of threedomains: a signal peptide of 29 amino acids, a propeptide of 17 aminoacids, which is required for γ-carboxylation of glutamic acid residues,and a mature Factor IX protein of 415 amino acid residues. The Factor IXzymogen undergoes three types of post-translational modifications beforeit is secreted into the blood: a vitamin K-dependent conversion ofglutamic acid residues to carboxyglutamic acids, addition of hydrocarbonchains, and β-hydroxylation of an aspartic acid. Mature Factor IXprotein contains 12 γ-carboxylated glutamic acid (Gla) residues. Due tothe requirement of vitamin K by γ-carboxylase, Factor IX is one ofseveral vitamin K-dependent blood coagulation factors.

The activation of Factor IX is achieved by a two-step removal of theactivation peptide (Ala¹⁴⁶-Arg¹⁸⁰) from the molecule. Bajaj et al.,“Human factor IX and factor IXa,” in METHODS IN ENZYMOLOGY (1993). Thefirst cleavage is made at the Arg¹⁴⁵-Ala¹⁴⁶ site by either Factor XIa orFactor VIIa/tissue factor. The second, and rate limiting cleavage ismade at Arg¹⁸⁰-Val¹⁸¹. The activation pathways involving Factor XIa andFactor VIIa/tissue factor are both calcium-dependent. However, theFactor VIIa/tissue factor pathway requires tissue factor that isreleased from damaged endothelial cells. Activated human Factor IX thusexists as a disulfide linked heterodimer of the heavy chain and lightchain. For full biological activity, human Factor IX must also have thepropeptide removed and must be fully γ-carboxylated. Kurachi et al.,Blood Coagulation and Fibrinolysis 4:953 (1993).

Factor IX is the precursor of a serine protease required for bloodclotting by the intrinsic clotting pathway. Defects in Factor IXsynthesis result in hemophilia B (or Christmas disease), an X-linkeddisorder that occurs in about one in 30,000 males. Patients withhemophilia B are treated with Factor IX obtained from pooled plasma fromnormal individuals. Martinowitz et al., Acta Haematol 94 (Suppl. 1):35(1995). Such Factor IX preparations, however, may be pyrogenic and maybe contaminated with pathogenic agents or viruses. Accordingly, it wouldbe advantageous to develop a means to prepare purified Factor IX thatdid not require extraction from human plasma.

In the past, therapeutic proteins have been produced in E. coli.However, limitations in secretion and post-translational modificationwhich occur in all living cells has rendered recombinant proteinproduction a highly species, tissue and cell specific phenomena. In anexample of recombinant FIX expression in mammalian cells, thepopulations of recombinant FIX produced in baby hamster kidney cells arenot the same protein products as FIX produced in Chinese hamster ovarycells (Busby et al., Nature 316:684-686 (1985); Kaufman et al., J. Biol.Chem. 261: 9622-9628 (1986)). These proteins have profound differencesin γ-carboxylation and propeptide removal and these differences havebeen established as being very important in determining biologicalactivity. Most importantly, only less than about 40 milliunits/hr/ml ofactive rFIX were detected in CHO cells even after coexpression of thepropeptide cleaving enzyme PACE, coexpression of the carboxylase enzyme,and extensive gene amplification with methotrexate in an attempt toincrease expression level and activity (Wasley et al. J. Biol. Chem.268: 8458-8465 (1993); Rehemtulla et al., Proc. Natl. Acad. Sci. (USA),90: 4611-4615 (1993)). Researchers concluded that multiple limitationsin the secretion of active rFIX exist in mammalian cells (Rehemtulla etal., 1993) and that the problem of gene transcription was secondary andindeed trivial with respect to post-translational processing ofbiologically active rFIX in mammalian cells. Thus, FIX mRNA splicing isa species specific effect occurring in mice and perhaps sheep, but notpigs. Although one might hypothesize that a FIX could be expressed, onecould not predict with any certainty whether such product would be aclinically acceptable, practical, recombinant therapeutic FIX productfor a given hemophiliac indication.

Production of recombinant Factor IX in mammalian cell culture (HepG2,mouse fibroblast, mouse hepatoma, rat hepatoma, BHK, CHO cells)repeatedly has been shown to be recalcitrant and cell-system specificwith respect to intracellular restrictions on secretion and proteolyticprocessing, post-translational modification, expression levels,biological activity, downstream recovery from production media, andsubstantiation of circulation half-life (Busby et al., (1985); de laSalle et al. Nature 316: 268-270 (1985); Anson et al., Nature 315:684-686 (1985); Rehemtulla et al., 1993; Wasley, et al., (1993); Kaufmanet al., (1986); Jallat et al., EMBO J. 9: 3295-3301 (1990)).Importantly, the aforementioned works concluded that nontrivialimprovements in these combined criteria are needed if a practicalprophylactic FIX therapeutic product is to be made available from anyrecombinant mammalian cell production source. For example, attempts toincrease the specific activity of rFIX produced by CHO cells byrectifying problems with under-carboxylation by co-expression of thevitamin K-dependent carboxylase enzyme resulted in no improvement inγ-carboxylation or biological activity (Rehemtulla et al., (1993),implying that multiple rate limitations in this post-translationalmodification exist.

Similar difficulties in the production of significant amounts ofbiologically active rFIX in the mammary epithelial cells of transgenicanimals also has been documented in the literature. AlthoughWO-A-90/05188 and WO-A-91-08216 predict that production of rFIX shouldbe possible in their production systems, no data are presented inWO-A-91-08216, and only very low levels of secreted rFIX (25 ng/ml) withno biological activity were reported in transgenic sheep in WO-A90/05188 and in related publications (Clark et al., Bio/Technology 7:487-4992 (1989)). Higher expression levels have recently been reportedin the milk of sheep (5 μg/ml), but again, the product had no biologicalactivity (Colman, IBC Third International Symposium on ExploitingTransgenic Technology for Commercial Development, San Diego, Calif.(1995)). This demonstrates that the polypeptides produced inWO-A-90/05188, Clark et al. (1989), and Colman (1995) were a differentspecies than native human FIX with dissimilar biological activity tohuman FIX, and could never be used for therapeutic purposes. Work byClark et al. (1992) stated that problems in synthesis of rFIX in themammary gland of transgenic mice was the result of aberrant splicing ofthe rFIX mRNA in the 3′ untranslated region. Correction of the aberrantsplicing in transgenic mice has been demonstrated (Yull et al. Proc.Natl. Acad. Sci. USA 92: 10899-10903 (1995); Clark, et al. (1989), WO95/30000)), resulting in higher expression levels (up to 61 μg/ml) withabout 40% biological active material. However, this aberrant splicingphenomenon appears to be species- and tissue-specific in the mousemammary gland; other reports with the 3′ UTR sequences in CHO cell linesand in the liver of transgenic mice specifically show no evidence ofaberrant splicing (Kaufman et al., (1986); Jallat et al., (1990)). Inaddition, no evidence was reported for aberrant mRNA splicing of FIXtranscripts with 3′ UTR sequences in a human hepatoma cell line (de laSalle et al., (1985)), a mouse fibroblast cell line (de la Salle et al.,(1985)), a rat hepatoma cell line (Anson et al., 1985)), or a BHK cellline (Busby et al., (1985)). No data are presented to justify theprediction that the altered transgene of WO95/30000 will necessarilyimprove the secretion and biological activity of rFIX in the milk oftransgenic livestock or any other cell line. Therefore the claimspresented in WO 95/30000 are purely speculative and are limited to themammary gland of transgenic mice.

The stability of the rFIX product in the milk of transgenic livestockduring upstream and downstream processing is a critical issue for theproduction of a practical therapeutic. Data presented in Clark et al.(1989) showed that Clark's method of downstream recovery of what littlerFIX was in the milk of their transgenic sheep was not reproducible: inone of the preparations, a significant amount of rFIX wasproteolytically activated. The infusion of activated FIX (FIXa) into apatient is fatal (Kingdon et al., Thrombosis, Diathes. Haemorrh.(Stuttg.) 33: 617 (1975)). FIX can be activated by FXI and/orFVIIa/Tissue factor complex in the presence of calcium and phospholipids(Kurachi et al., Blood Coagulation and Fibrinolysis 4: 953-974 (1993)).Milk is a medium containing calcium and phospholipid surfaces. Inaddition, there is extensively conserved homology between mammalianblood coagulation factors, especially between porcine FXI and human FXI(Mashiko and Takahashi, Biol. Chem. Hoppe-Seyler 375: 481-484 (1994)).Detectable levels of porcine FVII(a) and FXI(a) in the milk ofnontransgenic pigs, and elevated levels of FVII(a) and FXI(a) in themilk of a pig with mastitic milk have been measured. Thus, one couldpredict that the recovery of a useful unactivated rFIX produced in themilk of transgenic livestock will be very sensitive to the health of themammary gland (i.e., no subclinical or clinical mastitis), to themilking procedure (i.e., no tissue damage), to pretreatment of the milkimmediately after collection, to storage of the milk before processing,and to the purification and formulation process itself. One would alsopredict that the undesirable in vivo activation of rFIX also can beminimized by the coexpression of inhibitors to FVIIa/TF such as theTissue Factor Pathway Inhibitor (TFPI) protein, also called LACI, or thehybrid protein FX-LACI which is also a known inhibitor to FVIIa/TF.Although specific inhibitors of FXIa have not been identified, a similarapproach can be made for neutralizing FXIa activation by coexpression ofanalogues of polypeptide substrates of FXIa similar to those that arecommercially available for amidolytic assays. Yet another strategy maybe to overexpress rFIX at very high levels (>1 g/1 milk) such that theFIX activating enzyme is extremely limiting. Otherwise, steps must betaken immediately after milk collection to minimize activation. Theseinclude, but are not limited to, chelation of calcium (e.g., addition ofEDTA), phospholipid removal, adjustment of pH, storage in ultra-lowfreezers, controlled thawing procedures, addition of proteaseinhibitors, and purification procedures that maintain minimal activationconduciveness. If activated rFIX still persists in the purified product,removal can be facilitated by lectin chromatography (N-linkedcarbohydrate moieties exist only in the activation peptide),immunoaffinity chromatography using a Mab directed to the activationpeptide, or by metal ion induced precipitation techniques that canselect for the differences in molecular stability of unactivated vs.activated FIX. Because of these inherent difficulties in production ofactive FIX at sufficiently high levels in mammalian cells and transgeniclivestock, gene therapy has been cited as perhaps a more practical wayof achieving a prophylactic therapeutic rather than recombinanttechnology (Kurachi et al., (1993); Kay et al., Proc. Natl. Acad. Sci.USA 91: 2353-2357 (1994)); Fallaux et al., Thromb-Haemost. 74: 266-73(1995)). This is certainly a profound reality because it specificallyteaches a product suitable for FIX prophylaxis has not yet been foundusing recombinant production in mammalian cells, even those that havebeen shown to express active FIX, albeit at low levels. The bestrecombinant FIX cell production system made from CHO cells is producedat low secretion levels (Rehemtulla et al., (1993)) and is in fact notsuitable for prophylaxis. Furthermore, the data have shown that thehomologous plasma proteins FIX and protein C all have very different,cell-specific restrictions on post-translational processing, proteolyticprocessing, and secretion which preclude on a protein-specific basis thepredictability of high expression levels, biological activity,downstream recovery from production media, and predictable circulationhalf-life (Grinnell et al., “Native and Modified recombinant humanprotein C: function, secretion, and postranslational modifications,” InProtein C and Related Anticoagulants, eds. D. F. Bruley and Drohan29-63, Gulf Publishing Co., Houston, Tex. (1990); Yan et al., Trends inBiochem. Sci. (1989); Busby et al., (1985)).

Therefore, a need still exists for a means to obtain significant amountsof purified Factor IX from a source other than human plasma. A need alsoexists for a practical means for producing in mammalian cells rFIX,which is suitable as a treatment for hemophilia B.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide amethod for producing a transgenic animal that secretes biologicallyhuman active Factor IX into its milk.

It is a further object of this invention to provide a transgenic animalthat produces at least 100 μg of human Factor IX per milliliter of milk.

These and other objects are achieved, in accordance with one embodimentof the present invention by the provision of a transgenic non-humanmammal containing an exogenous DNA molecule stably integrated in itsgenome.

A non-human transgenic mammal containing an exogenous DNA moleculestably integrated in its genome, wherein said exogenous DNA moleculecomprises:

-   -   (a) 5′ regulatory sequences of a mammary gland-specific gene        including a promoter;    -   (b) a Factor IX-encoding DNA sequence that encodes a signal        sequence, a Factor IX pro-sequence and a Factor IX sequence in a        5′ to 3′ direction, wherein said signal sequence is effective in        directing the secretion of said Factor IX into the milk of said        transgenic mammal and wherein said Factor IX sequence lacks at        least a portion of the complete or the complete 5′-untranslated        and 3′-untranslated regions of the Factor IX gene; and    -   (c) 3′ regulatory sequences from a mammary gland-specific gene        or 3′ regulatory sequences active in a mammary gland;

wherein said 5′ and said 3′ regulatory sequences are operatively linkedto said Factor IX-encoding DNA sequence.

Mammary gland-specific promoters that are useful in the presentinvention are selected from the group consisting of short whey acidicprotein (WAP) promoter, long WAP promoter, short α-casein promoter,short β-casein promoter, short kappa-casein promoter, long α-caseinpromoter, long β-casein promoter, long kappa-casein promoter,α-lactalbumin promoter and β-lactoglobulin promoter.

Non-human transgenic mammals which are contemplated by the presentinvention are selected from the group consisting of mice, rats, rabbits,pigs, sheep, goats and cows.

It is a further object to provide a process for producing Factor IX byproviding a non-human transgenic mammal having integrated into itsgenome an exogenous DNA molecule, wherein said exogenous DNA moleculecomprises:

-   -   (a) providing a non-human transgenic mammal having integrated        into its genome an exogenous DNA molecule, wherein said        exogenous DNA molecule comprises: (1) 5′ regulatory sequences of        a mammary gland-specific gene including a promoter; (2) a Factor        IX-encoding DNA sequence that encodes a signal sequence, a        Factor IX pro-sequence and a Factor IX sequence in a 5′ to 3′        direction, wherein said signal sequence is effective in        directing the secretion of said Factor IX into the milk of said        transgenic mammal and wherein said Factor IX sequence lacks at        least a portion of the complete or the complete 5′-untranslated        and 3′-untranslated regions of the Factor IX gene; and (3) 3′        regulatory sequences from a mammary gland-specific gene or 3′        regulatory sequences active in a mammary gland; wherein said 5′        and said 3′ regulatory sequences are operatively linked to said        Factor IX-encoding DNA sequence;    -   (b) allowing said DNA sequences encoding said Factor IX to be        expressed and said Factor IX to be secreted into the milk of        said transgenic mammal;    -   (c) collecting said milk from said mammal; and    -   (d) isolating said Factor IX from said milk.

It is a further object to provide a method of treating hemophilia Busing the Factor IX produced by the transgenic mammal, described above.Treating involves administration of the Factor IX of the invention and apharmaceutically acceptable carrier to a hemophilia B patient.

It is a further object of the invention to provide an in vitro cultureof mammary gland cells that produce Factor IX. Another object of theinvention is to provide a method of treating hemophilia B by implantingsuch Factor IX mammary gland cells into a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D schematically depict the construction of a chimeric FactorIX construct. Specifically, FIG. 1A shows the construction of pWAP4.FIG. 1B shows the production of pUCFIX. FIG. 1C shows the introductionof human FIX cDNA into pWAP4. FIG. 1D shows the production of pUCWAPFIX.FIX cDNA was modified by PCR in order to introduce KpnI sites on the 3′and 5′ ends. Using FIX cDNA as a template, PCR primers humFIX5′KpnI andhumFIX3′KpnI, as shown in Table 1, below, were used to produce FIX cDNAwith KpnI sites on both ends. Modified cDNA may be easily into a“cassette vector” for constructing a chimeric gene.

FIG. 2 shows the detection of recombinant Factor IX in transgenic pigmilk using western blot analysis.

FIGS. 3A-3C show the production of the pUCWAP6 “cassette vector.”Specifically, FIG. 3A shows the production of pUCNotI. FIG. 3B shows theproduction of pUCWAP5 and the production of a fragment that contains thepUCNotI vector sequence flanked by mWAP3′UTR. FIG. 3C shows theproduction of pUCWAP6.

FIG. 4 shows the production of plasmid pUCWAP6FIX.

DETAILED DESCRIPTION 1. Overview

As discussed above, a method for producing significant quantities ofFactor IX in transgenic animals has been elusive. Yull et al., Proc.Nat'l Acad. Sci. USA 92:10899 (1995), showed that correction of acryptic RNA splice site increases the amount of Factor IX synthesized bytransgenic animals. In these studies, one transgenic mouse line producedabout 27 μg of biologically active Factor IX per milliliter of milk,although, Factor IX levels of individual mice of the line varied. Yullet al. speculated that the variation was probably due to epigeneticinstability.

In contrast, the studies presented herein show that transgenic pigs cansynthesize and secrete high levels (100-200 μg/ml milk) of biologicallyactive recombinant human Factor IX in milk. Based on reduced andnonreduced SDS PAGE, the majority of the recombinant human Factor IXpopulation appears to be a single chain polypeptide having apost-translationally modified structure similar to human Factor IX. Therecombinant human Factor IX secreted into pig milk is biologicallyactive and is able to initiate clotting in Factor IX-deficient humanplasma. This is the first reported production of high levels of fullyactive, sufficiently γ-carboxylated, recombinant human Factor IX in themilk of transgenic livestock.

2. Methods for Producing Transgenic Animals

Notwithstanding past failures to express recombinant human Factor IXwith suitably high activity in several different expression systems, thepresent invention provides methods for obtaining recombinant Factor IXcharacterized by a high percentage of active protein from the milk oftransgenic animals. As used herein, the term “animal” denotes allmammalian animals except humans. It also includes an individual animalin all stages of development, including embryonic and fetal stages. A“transgenic” animal is any animal with cells that contain geneticinformation received, directly or indirectly, by deliberate geneticmanipulation at the subcellular level, such as by microinjection orinfection with recombinant virus.

The genetic information to be introduced into the animal is preferablyforeign to the species of animal to which the recipient belongs (i.e.,“heterologous”), but the information may also be foreign only to theparticular individual recipient, or genetic information alreadypossessed by the recipient. In the last case, the introduced gene may bedifferently expressed than is the naturally occurring, or “native,”gene.

The language “germ cell line transgenic animal” refers to a transgenicanimal in which foreign DNA has been incorporated into a germ line cell,therefore conferring the ability to transfer the information tooffspring. If such offspring, in fact, possess some or all of thatinformation, then they, too, are transgenic animals.

The transgenic animals of this invention are other than human,including, but not limited to farm animals (pigs, goats, sheep, cows,horses, rabbits and the like), rodents (such as mice), and domestic pets(for example, cats and dogs). Livestock animals such as pigs, sheep,goats and cows, are particularly preferred.

Preferably, a transgenic animal of the present invention is produced byintroducing into single cell embryos appropriate polynucleotides thatencode human Factor IX, or fragments or modified products thereof, in amanner such that these polynucleotides are stably integrated into theDNA of germ line cells of the mature animal, and are inherited in normalMendelian fashion.

In accordance with the invention, DNA molecules can be introduced intoembryos by a variety of means to produce transgenic animals. Forinstance, totipotent or pluripotent stem cells can be transformed bymicroinjection, calcium phosphate mediated precipitation, liposomefusion, retroviral infection or by other means. The transformed cellscan then be introduced into embryos and incorporated therein to formtransgenic animals. In a preferred method, developing embryos can beinfected with retroviral vectors and transgenic animals can be formedfrom the infected embryos. In the most preferred method, however, theDNA molecules of the invention are injected into embryos, preferably atthe single-cell stage, which are allowed to develop into maturetransgenic animals. However, the present invention is not limited tothis preferred method but other methods of making transgenic animals canbe used as described, for example, in Transgenic Animal Generation andUse by L. M. Houdebine, Harwood Academic Press, 1997. Transgenic animalsalso can be generated using methods of nuclear transfer or cloning usingembryonic or adult cell lines as described for example in Campbell etal., Nature 380: 64-66 (1996) and Wilmut et al., Nature 385: 810-813(1997). Further a technique utilizing cytoplasmic injection of DNA canbe used as described in U.S. Pat. No. 5,523,222.

Factor IX-producing transgenic animals can be obtained by introducing achimeric construct comprising Factor IX-encoding sequences. Analternative method of producing transgenic animals is to introduce aFactor IX chimeric construct with a second construct that may providehigher expression more frequently than that observed with the use ofFactor IX constructs alone. As described herein, such doubly-transgenic,or “bigenic,” animals have native WAP genomic sequences that areinjected as separate constructs to be concatenated in vivo as additionalflanking sequences to the target Factor IX cDNA construct.

Methods for obtaining transgenic animals are well-known. See, forexample, Hogan et al., MANIPULATING THE MOUSE EMBRYO, (Cold SpringHarbor Press 1986); Krimpenfort et al., Bio/Technology 9:88 (1991);Palmiter et al., Cell 41:343 (1985); Kraemer et al., GENETICMANIPULATION OF THE EARLY MAMMALIAN EMBRYO, (Cold Spring HarborLaboratory Press 1985); Hammer et al., Nature 315:680 (1985); Wagner etal., U.S. Pat. No. 5,175,385; Krimpenfort et al., U.S. Pat. No.5,175,384, Jänne et al., Ann. Med. 24:273 (1992), Brem et al., Chim.Oggi. 11:21 (1993), Clark et al., U.S. Pat. No. 5,476,995, herebyincorporated by reference.

3. Construction of Chimeric Genes

Suitable Factor IX-encoding DNA used for producing transgenic animalscan be obtained using human liver tissue as a source for cloning thehuman Factor IX gene. The DNA coding for Factor IX can be fused, inproper reading frame, with appropriate regulatory signals, as describedin greater detail below, to produce a chimeric construct which is thenamplified, for example, by propagation in a bacterial vector, accordingto conventional practice.

The amplified construct is thereafter excised from the vector andpurified for use in microinjection. The purification is preferablyaccomplished by means of high performance liquid chromatography (HPLC),which removes contamination of the bacterial vector and frompolysaccharides typically present when other techniques, such asconventional agarose electroelution, are used. The preferred HPLC methodentails sorbing the construct onto an anion-exchange HPLC support andselectively eluting the construct from the support, preferably with anaqueous sodium chloride solution, thereby to eliminate contaminationfrom the vector. Elution also may be effected by other means, such as apH gradient.

Alternatively, the excised construct can be purified byultracentrifugation through an aqueous sucrose or sodium chloridegradient, gel electroelution followed by agarose treatment and ethanolprecipitation, or low pressure chromatography.

Since it is preferable that the construct have the minimum amount ofimpurities, more than one cycle of HPLC or other purification isadvantageous. In particular, the use of HPLC-purified DNA formicroinjection, as described above, allows for remarkably hightransformation frequencies, on the order of 20% or more, for example, inmice and pigs.

DNA constructs useful in the present invention provide a DNA sequenceencoding Factor IX, preferably human Factor IX, operably linked to allthe cis-acting signals necessary for mammary tissue specific expressionof Factor IX, post-translational modification of Factor IX, secretion ofFactor IX into milk, and full biological activity of Factor IX. Althoughthe present invention preferably entails the use of DNA constructs thatproduce the desired or native human Factor IX per se, the desiredprotein also may be produced as a fusion protein containing anotherprotein. For example, the desired recombinant protein of this inventionmay be produced as part of a larger recombinant protein in order tostabilize the desired protein or to make its purification from milkfaster and easier. The fusion partners then are separated chemically orenzymatically, and the desired protein isolated.

Methods for obtaining human Factor IX-encoding DNA molecules andnucleotide sequences of human Factor IX gene and cDNA are provided, forexample, by Kurachi et al., Proc. Nat'l Acad. Sci. USA 79:6461 (1982),Choo et al., Nature 299:178 (1982), Anson et al., EMBO J. 3:1053 (1984),Brownlee et al., international publication No. WO 84/00560, Yull et al.,Proc. Nat'l Acad. Sci. USA 92: 10899 (1995), Clark, internationalpublication No. WO 95/30000, and Meulien, U.S. Pat. No. 5,521,070(1996). Human Factor IX probes also can be obtained from the AmericanType Culture Collection, Rockville, Md. (e.g., ATCC Nos. 61385, 79588,79602, or 79610).

Alternatively, Factor IX-encoding DNA molecules may be obtained bysynthesizing the genes with mutually priming long oligonucleotides. See,for example, Ausubel et al., supra, at pages 8.2.8 to 8.2.13; Wosnick etal., Gene 60:115 (1987). Moreover, the polymerase chain reaction can beused to synthesize DNA fragments as large as 1.8 kilobases in length.Bambot et al., PCR Methods and Applications 2:266 (1993).

Suitable Factor IX-encoding DNA molecules include genomic orcomplementary DNA molecules that encode naturally occurring Factor IX.In a preferred embodiment, DNA molecules encoding human Factor IX areemployed, including cDNA and genomic DNA molecules. However, the presentinvention discloses that a cDNA based construct as described herein canbe successfully used for the expression of human Factor IX atcommercially useful levels. Particularly a cDNA based constructcontaining 5′ regulatory sequences of a mammary gland specific geneincluding a promoter, a Factor IX-encoding DNA sequence as describedherein, and 3′ regulatory sequences from a mammary gland-specific geneor 3′ regulatory sequences active in a mammary gland is preferred.Factor IX-encoding DNA molecules from other species may also be used,such as the Factor IX encoded by rats, pigs, sheep, cows andchimpanzees.

It also will be appreciated that the Factor IX cDNA fragment describedherein can be modified using recombinant DNA techniques to obtainfunctionally equivalent molecules. For example, 3′ or 5′ portions of theFactor IX gene can be added, or completely deleted, or a few bases ateither end may be removed. Introns can be removed or added, or portionsof one or more introns can be deleted. Additional nucleotide sequencescan be inserted into them. The sequences of the introns can be altered.Exons can be modified in accordance with the discussion of modifiedFactor IX molecules set forth below. Most modified forms of thepreferred Factor IX cDNA fragment will not be significantly changed intheir ability in transgenic animals to engender the production ofmilk-born Factor IX. In one embodiment, the Factor IX encoding portionof the gene lacks the complete 5′-untranslated and 3′-untranslatedregions of the native Factor IX gene. Thus, these substantially similarfragments will be equivalent in the invention to the particularlydisclosed Factor IX cDNA fragment.

A 5′-untranslated region that is not the 5′-untranslated region of theFactor IX gene can be included in the present DNA Factor IX constructs,particularly the 5′-untranslated region of the mouse WAP gene. Likewisea 3′-untranslated region that is not the 3′-untranslated region of theFactor IX gene, particularly the 3′-untranslated region of the mouse WAPgene.

Further, the Factor IX-encoding DNA molecule can also comprise a5′-untranslated region located 5′ from the signal sequence DNA, and a3′-untranslated region located 3′ from the Factor IX coding sequence.

Additional useful modifications of Factor IX include those that alterpost-translational modifications, size or active site, or that fuse thisprotein or portions thereof to another protein. Such modifications canbe introduced into the protein by techniques well known in this art,such as by synthesizing modified genes by ligation of overlappingoligonucleotide or introducing mutations into the cloned genes by, forexample, oligonucleotide-mediated mutagenesis. See, generally, Ausubelet al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages 8.0.3-8.5.9(1990); McPherson (ed.), DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRLPress (1991).

The examples described herein demonstrate that a transgenic animal canbe produced that synthesizes a sufficiently γ-carboxylated, biologicallyactive Factor IX in mammary tissue. Accordingly, the basic methods ofthe present application can be used to obtain transgenic animals thatproduce other vitamin K-dependent blood coagulation factors, such asFactor II, Factor VII, Factor X, or the anticoagulation protein, ProteinS. DNA molecules encoding these proteins can be obtained by standardmethods. See, for example, Pollak et al., J. Biol. Chem. 271: 1738(1996), which describes the characterization of the Factor VII gene,which is located on chromosome 13 just 2.8 kilobase pairs 5′ to theFactor X gene.

The cis-acting regulatory regions useful in the invention include thepromoter that drives expression of the Factor IX gene. Promotersparticularly useful in the invention are “active” in mammary tissue inthat the promoters are more active in mammary tissue than in othertissues under physiological conditions where milk is synthesized. Mostpreferred are promoters that are both specific to and efficient inmammary tissue. By “efficient” it is meant that the promoters are strongpromoters in mammary tissue that can support the synthesis of largeamounts of protein for secretion into milk. Among such promoters, highlypreferred are the short and long whey acidic protein (WAP), short andlong α, β and kappa casein, α-lactalbumin and β-lactoglobulin (“BLG”)promoters.

Promoters may be selected on the basis of the protein compositions ofmilk from various species. For example, the WAP and BLG promoters areparticularly useful with transgenic rodents, pigs and sheep. The rodentWAP short and long promoters have been used to express the rat WAP gene,the human tissue-type plasminogen activator gene and the CD4 gene, whilethe sheep BLG promoter has been used to express the sheep BLG gene, thehuman alpha-1-antitrypsin gene and the human Factor IX gene. See, forexample, Paleyanda et al., 1991, above, and Clark et al., TIBTECH 5: 20(1987). Preferred promoters include the rodent casein and WAP promoters,and the casein, α-lactalbumin and BLG promoters from porcine, bovine,equine and ovine (pigs, sheep, goats, cows, horses), rabbits, rodentsand domestic pets (dogs and cats). The genes for these promoters havebeen isolated and their nucleotide sequences have been published. See,for example, Clark et al. (1987), above, and Henninghausen, ProteinExpression and Purification 41: 3 (1990).

A useful promoter may be isolated by carrying out conventionalrestriction endonuclease and subcloning steps. A mouse WAP promoter,isolated as a 2.6 kb EcoRI-KpnI fragment immediately 5′ to the WAPsignal sequence, is preferred, although the “long” WAP promoter (the 5′4.2 kb Sau3A-KpnI promoter of the mouse WAP gene, or a fragment thereof)is also suitable for carrying out this invention. The publication ofPaleyanda et al., Transgenic Research 3: 335 (1994), for example,provides examples of a suitable short mouse WAP promoter (“2.5 kb mWAPpromoter”) and a long mouse WAP promoter (“4.1 kb mWAP promoter”). Pages336-339 of the Paleyanda publication are incorporated by reference. Alsosee, for example, Gordon et al., Bio/Technology 5: 1183 (1987); McKnightet al., “The Whey Acidic Protein,” in GENES, ONCOGENES, AND HORMONES:ADVANCES IN CELLULAR AND MOLECULAR BIOLOGY OF BREAST CANCER, Dickson etal. (eds.), pages 399-412 (Kluwer Academic Publishers 1991). Additionalregulatory sequences direct secretion of proteins into milk and/or otherbody fluids of the transgenic animal. In this regard, both homologousand heterologous regulatory sequences are useful in the invention.Generally, regulatory sequences known to direct the secretion of milkproteins, such as either signal peptides from milk or the nascent targetpolypeptide, can be used, although signal sequences can also be used inaccordance with this invention that direct the secretion of expressedproteins into other body fluids, particularly blood and urine. Examplesof such sequences include the signal peptides of secreted coagulationfactors including signal peptides of Factor IX, protein C, andtissue-type plasminogen activator.

Among the useful sequences that regulate transcription, in addition tothe promoters discussed above, are enhancers, splice signals,transcription termination signals, polyadenylation sites, bufferingsequences, RNA processing sequences and other sequences which regulatethe expression of transgenes. Particularly useful in this regard arethose sequences that increase the efficiency of the transcription of thegenes for Factor IX in the mammary gland or other cells of thetransgenic animals listed above. Preferred are transcription regulatorysequences for proteins highly expressed in the mammary gland cells.

Preferably, the expression system or construct of this invention alsoincludes a 3′ untranslated region downstream of the DNA sequenceencoding the desired recombinant protein, or the 3′ untranslated regionof the milk protein gene or the milk protein gene with its 3′untranslated region, any of which can be used for regulation. Thisregion can increase expression of the transgene. This region apparentlystabilizes the RNA transcript of the expression system and thusincreases the yield of the desired protein. Among the 3′ untranslatedregions useful in this regard are sequences that provide a poly Asignal.

For expression of Factor IX, it is preferred that the 3′ untranslatedregion is not obtained from the native human Factor IX gene. Suitableheterologous 3′-untranslated sequences can be derived, for example, fromthe SV40 small t antigen, the casein 3′ untranslated region, or other 3′untranslated sequences well known in this art. Preferably, the 3′untranslated region is derived from a milk-specific protein, such as theWAP protein. The stabilizing effect of this region's poly A transcriptis important in stabilizing the mRNA of the expression sequence.Ribosome binding sites are also important in increasing the efficiencyof expression of Factor IX. Likewise, sequences that regulate thepost-translational modification of Factor IX are useful in theinvention.

In a particularly preferred embodiment, the transgenes of the inventiongenerally consist of WAP milk protein regulatory sequences upstream anddownstream flanking the Factor IX cDNA/signal peptide sequences. Anative 5′-WAP regulatory sequence ending in an accessible restrictionsite immediately before/at the ATG codon may be ligated to therestriction sites that occur at the ATG of translatable sequences withno linker sequences derived from the chains of human Factor IX. Each ofthe combined 5′-regulatory and Factor IX translatable sequences endingin a particular restriction site may then be ligated to a correspondingrestriction site which occurs at the beginning of the 3′-untranslatedregion of WAP and adjoining WAP 3′-flanking region. This constructionmotif enables native 5′-regulatory and 3′-untranslated region of themilk protein genes to be immediately juxtaposed without interveningsequences. Particular restriction sites at the ends of all constructsmay be selected in order to facilitate concatenation of constructs intoa single domain within the animal genome.

Thus, in accordance with the present invention a DNA molecule thatencodes Factor IX is operably linked to cis-acting regulatory sequenceswhich allow for efficient expression of Factor IX in milk. The resultingchimeric DNA is introduced into a mammalian embryo, where it integratesinto the embryonic genome and becomes part of the heritable geneticendowment of all the cells, including the germ line cells, of the adultwhich develops from the embryo. The Factor IX which is expressed in themammary tissue and secreted into the milk of a transgenic mammalobtained in this manner displays a surprisingly high percentage ofactive protein, as measured by enzymatic and coagulation-inhibitionassays which are conventionally employed to detect Factor IX activity,such as ELISAs, chromogenic activity assays and coagulation inhibitionassays.

4. Isolation of Factor IX from the Milk of Transgenic Animals

Obtaining milk from a transgenic animal according to the presentinvention is accomplished by conventional means. See, for example,McBurney et al., J. Lab. Clin. Med. 64:485 (1964); Velander et al., ProcNat'l Acad. Sci. USA 89:12003 (1992). Factor IX, or fragments thereof,can be isolated and purified from milk or urine by conventional meanswithout deleteriously affecting activity. A preferred method ofisolation from milk consists of a combination of anion exchange andimmunochromatography, cryoprecipitations, zinc ion-induced precipitationof either whole milk or milk whey (defatted milk) proteins. See, forexample, Bringe et al., J. Dairy Res. 56:543 (1989).

Milk is known to contain a number of proteases that have the potentialto degrade foreign proteins. These include an alkaline protease withtryptic and chymotryptic activities, a serine protease, achymotrypsin-like enzyme, an aminopeptidase and an acid protease. Clarket al. (1987) above. It may be desirable, therefore, to protect newlysecreted Factor IX, or fragments thereof, against proteolyticdegradation. Such precautions include rapid processing of the milk aftercollection and addition to the milk of well known inhibitors ofproteolysis, such as are listed in SIGMA CHEMICAL CO. CATALOG (1993edition) at page 850.

Thus, in one embodiment, the transgenic mammal of the present inventionproduces active human Factor IX. For instance, in one embodiment whereinsaid mammal is a pig, such pig secretes from about 100 to about 220 μgof active human Factor IX per milliliter milk. In another embodiment,such pig secretes from about 100 to about 185 μg of active human FactorIX per milliliter milk, from about 100 to about 170 μg of active humanFactor IX per milliliter of milk, from about 135 to about 220 μg ofactive human Factor IX per milliliter of milk or from about 145 to about220 μg of active human Factor IX per milliliter of milk, as set forthbelow.

Factor IX produced from the transgenic mammal according to the inventionhas a specific activity which is at least about 5 to 200 percent greaterthan the specific activity of human Factor IX isolated from humanplasma, as determined by an activated partial thromboplastin clottingtime assay. In another embodiment, the specific activity of Factor IXproduced by the transgenic mammal of the invention is at least about 10to 100 percent greater, at least about 15 to 50 percent greater or atleast about 15 to about 46 percent greater than the specific activity ofhuman Factor IX isolated from human plasma.

In another embodiment, the invention relates to an in vitro culture ofmammary gland cells explanted from the transgenic mammal of theinvention. Such cells are explanted and cultured in vitro, according tomethods well known to the skilled artisan. See e.g., U.S. Pat. No.5,580,781. In another embodiment, Factor IX is isolated and purifiedfrom the in vitro cell culture, according to methods well known to theskilled artisan.

5. Treatment Methods

In another embodiment, the present invention relates to a method oftreating hemophilia B using Factor IX produced by the transgenic mammalof the invention. Specifically, treatment includes the prevention oramelioration of the symptoms of hemophilia B in hemophilia B patients.Symptoms of hemophilia B include excessive bleeding upon injury,spontaneous bleeding, especially into weight-bearing joints, softtissues and mucous membranes. Repeated bleeding into joints results inhemarthroses, which causes painful crippling arthropathy thatnecessitates joint replacement. Hematomas in soft tissues may result in“pseudo” tumors composed of necrotic coagulated blood. Such blood canobstruct, compress or rupture into adjacent organs and can lead toinfection. Bleeding into gastrointestinal tract, central nervous system,intracranium or airway/retroperitoneal space can lead to death if notdetected. This, treatment according to the present invention includesthe prevention or amelioration of bleeding and the related side effectsfound in hemophilia B patients. This method involves administering to apatient having hemophilia B symptom, a hemophilia B symptom preventingor ameliorating amount of Factor IX produced by the transgenic mammal ofthe present invention. Administration may be accomplished by any methodknown to the skilled artisan. For instance, the treatment of the abovedescribed symptoms may consist of intravenous replacement therapy withFactor IX concentrates. Treatment of major bleeding episodes may be bybolus injection of concentrate. However, as described above, tissuedamage may remain even after prompt detection and treatment.Prophylactic treatment is recommended to prevent pain and debilitation.Upon injection, 50% of Factor IX, according to the invention, isimmediately bound to vascular endothelial cells and/or diffuses into theextravascular space. The remaining 50% has a half life in circulation ofapproximately 24 hours. These infusion kinetics result in the need forinjections about once to twice per week to maintain minimal therapeuticlevels in the plasma.

Another embodiment of the invention relates to pharmaceuticalcompositions comprising the Factor IX of the present invention. Suchpharmaceutical composition preferably is Factor IX produced by the abovedescribed transgenic animal and a pharmaceutically acceptable carrier.For instance, such pharmaceutical composition may be a stable liquidformulation of the Factor IX of the invention that can be administeredby continuous infusion to provide a constant circulating level of thecoagulation factor.

The Factor IX produced by the transgenic animal of the present inventionmay be concentrated and sold in lyophilized form, according to methodswell known to the skilled artisan. For instance, the Factor IX of thepresent invention which has been lyophilized may be reconstituted withsterile water for injection (WFI) and delivered in a composition of:0.01 moles/liter histidine, pH 7.05; 0.066 moles/liter sodium chloride;3% mannitol. In another embodiment, lyophilized Factor IX isreconstituted in sterile WFI and delivered in a composition thatincludes: 0.04 units heparin/unit FIX; 1 milligram dextrose/unit FactorIX. To avoid repeated invasive treatments as is found with the currenttherapies for prophylaxis, stabilities of at least 30 days at 37° C. andat least 365 days at 4° C. are preferred. The present invention providessignificant stability over that of these preparations reconstituted.

This skilled artisan would know of other suitable formulations for theFactor IX of the present invention. See, for instance, AlphaNine byTherapeutic Corporation, Los Angeles, Calif., and Bebulin V H, byImmuno, Vienna, Austria. Of course, any formulations according to thepresent invention are highly purified and free of viruses, prions,blood-group antibodies, immune complexes and phospholipids.

Dosages or amounts that prevent or ameliorate the symptoms of hemophiliaB are necessarily dictated by the clinical picture and severity of thedisease. Because there is so much variability between patients and theirclinical conditions, monitoring of coagulation function is essential induring any therapy using the Factor IX of the invention. As a rule, oninitial treatment, one unit of Factor IX per kg body weight gives a meanrise in Factor IX activity of about 0.5-1%, on continuation therapy, themean rise is about 1-1.5% Examples of dosages for long term prophylaxisof symptoms of hemophilia B are about 18-30 IU/kg (1× weekly) or about9-15 IU/kg (2× weekly). Dosages also will vary depending upon thepurpose of the treatment. For instance, where a hemophilia B patient hashad surgery, it may be desirable to raise Factor IX levels in suchpatients by 30 to 50% following the week of surgery. For dentalextractions, the Factor IX levels may need to be raised to 50%immediately prior to the surgery. Mild to moderate hemorrhages may betreated with a single administration of the Factor IX of the inventionto raise Factor IX levels to 20 to 30%. In the even to more serioushemorrhages, it may be desirable to raise Factor IX levels to 30 to 50%and infusions may be required daily. Again, those of skill in the artwould know how to adjust the amount and frequency of dosages of theFactor IX of the present invention depending upon the patient and theclinical setting.

In yet another embodiment, the invention relates to a method of treatinghemophilia B using Factor IX-producing cells that are explanted from thetransgenic mammal of the present invention. Such mammary gland cellsexpress Factor IX in vivo, thereby preventing or ameliorating thesymptoms of hemophilia B. This method is accomplished by using knowntechniques for gene therapy. See e.g., Debs, R. Proc. Natl' Acad. Sci.(USA) 89: 11277-11281 (1992), Legendre et al., Pharmaceutical Res. 9:1235-42 (1992). In one embodiment, Factor IX-producing cells removedfrom the transgenic mammal according to the invention are cultured in anin vitro culture system prior to transplantation into a human. Suchculture systems are well known to the skilled artisan. See e.g. U.S.Pat. No. 5,580,781. The cells are treated and then transplanted into thepatient in a manner so as to avoid rejection by the recipient. Suchmethods are known to the skilled artisan. See, for instance, U.S. Pat.No. 5,573,934, which teaches a method of encapsulating biologicalmaterial for use in vivo. Other techniques known to the skilled artisaninvolve placing the biological material in a chamber of animmunoisolation apparatus and for enhancing the vascular support for theimplanted material using immunomodulatory agents. See, U.S. Pat. No.5,569,462.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and are not intended to be limiting of the presentinvention.

Example 1 Preparation of a Human Factor IX Expression Vector forProduction of Transgenic Pigs

Generally, the entire murine WAP gene including 2.5 kb of 5′untranslated sequence and 3′ untranslated regions was cloned by standardmethods. See Campbell et al., Nucleic Acids Res. 12:8685 (1984). A cDNAfragment encoding human Factor IX was obtained and the 3′ untranslatedregion was deleted. Using standard methods, an expression vector wasconstructed that contained a mouse WAP promoter, isolated as a 2.6 kbEcoRI-KpnI fragment immediately 5′ to the WAP signal sequence, the humanFactor IX cDNA sequence lacking a 3′ untranslated region, and a 1.6 kbfragment of the 3′ untranslated region of the WAP gene. A secondexpression vector contained a 7.2 kb mouse WAP gene (EcoRI-EcoRI)fragment. Expression vectors were amplified by bacterial transformationand purified from bacterial cultures using standard methods. Routinerecombinant DNA techniques can be found, for example, in Sambrook etal., MOLECULAR CLONING, A LABORATORY MANUAL, Vol. 1-3 (Cold SpringHarbor Press 1989).

More specifically, a chimeric Factor IX construct was prepared, asfollows:

1. Preparation of a Chimeric Factor IX Construct Production of pWAP4“Cassette Vector”

Regulatory 5′ and 3′ flanking sequences of the mouse WAP gene were usedfor mammary specific expression. Specifically, a cassette vectorcontaining a mouse WAP promoter, defined as a 2.6. kb EcoRI-KpnIfragment immediately 5′ to the WAP signal sequence and a 1.5 kb fragmentof the 3′ untranslated region of the WAP gene was prepared. Theseregulatory sequences do not include coding and intragenic untranslatedsequences (introns) of the WAP gene.

The vector designated pWAP4 was derived from pWAPPC3 (C. Russell,dissertation “Improvement of Expression of Recombinant Human Protein Cin the Milk of Transgenic Mammal Using a Novel Transgenic Construct,”Virginia Technology Institute, Blacksburg, Va. (December 1993)) and wasdeveloped as follows: Using WAPPC3 as a template, PCR primers WAP3′S2(which contains a 5′KpnI site and is homologous to endogenous WAP rightafter the stop signal) and WAP3′A1, as shown in Table 1, below, wereused to produce a segment with KpnI and BamHI sites on either end. Thissegment was digested with KpnI/BamHI and ligated with the vectorcontaining the fragment from KpnI\BamHI digested pWAPPC3. The ligationmixture was used to transform E. coli DH5α cells by electroportationwith resultant colonies grown on LB ampicillin plates. Picked colonieswere grown up in TB ampicillin broth, plasmids isolated and cut withKpnI, BamHI or both and subjected to gel electrophoresis. Sequencing wasperformed using WAP3′A1 primer and judged as being correct. See FIG. 1A.

Production of Modified (Kpn I) FIX cDNA

The FIX cDNA (containing Kpn I sites located immediately before thestart sequence and after the stop sequence) was generated as a PCRfragment. Fragment production protocol is as follows: 100 μl totalvolume containing 200 μM dNTP's, 0.5 μM of each primer (humFIX5′KpnI andhumFIX3′KpnI, as shown in Table 1), 2.5 units Pfu polymerase and 30 ngof plasmid template (pMCDSFIX obtained from Prof. Darryl Stafford,Department of Biology, University of North Carolina, Chapel Hill, N.C.,USA), reaction mixture was subjected to 30 cycles of denaturation at 95°C. for 20 sec, annealing at 50° C. for 1 min and elongation at 75° C.for 5 min 45 sec. After cycling, the reaction mixture was subjected toblunting with T4 DNA polymerase for 10 min, EDTA concentration broughtup to 25 mM, heated to 65° C. for 15 min, and extracted withPhenol:Chloroform (1:1), precipitated with equal volumes of 95% ethanol,aspirated, and suspended in H₂O.

Ligation, Transformation and Sequencing

As is shown in FIG. 1B, the plasmid designated pUCFIX containing themodified (Kpn I ends) FIX cDNA was produced by digestion of both pUC18and the modified cDNA with Kpn I (per manufacturers instructions,Stratagene, La Jolla, Calif.) purification of digestion products byCHCl₃:Phenol (1:1) extraction, precipitation with equal volumes of 95%ethanol, aspiration and suspension in H₂O. Ligation of plasmid and cDNAwas per manufacturers instructions (Stratagene) using 125 ng of Kpn Idigested pUC18 and 125 ng of Kpn I digested modified cDNA. E. coli JM109was transformed by electroportation using ligation mixture and plated onLB ampicillin plates. Selected colonies were grown up in TB ampicillinbroth. Plasmid preparations from these colonies were analyzed byrestriction enzyme digestion (Kpn I) and gel electrophoresis. The entiresense strand of the cDNA was sequenced and found to be correct ascompared with FIXA sequences located in Genebank.

Introduction of FIX cDNA into pWAP4 “Cassette Vector” to Produce pWAPFIX

As shown in FIG. 1C, both pWAP4 and pUCFIX were digested with Kpn I inseparate reactions, subjected to gel electrophoresis and the appropriateplasmid fragments removed from the gel and ligated. E. coli JM109 wastransformed by electroportation using ligation mixture and plated on LBampicillin plates. Selected colonies were grown up in TB ampicillinbroth. Plasmid preparations from these colonies were analyzed byrestriction enzyme digestion (Kpn I) then gel electrophoresis. Clonespositive for the insert were subjected to PCR analysis using primersFIXS1 and WAP3′A1 to determine the correct orientation of the insert.

Production of pUCWAPFIX

As shown in FIG. 1D, the insert containing WAP promoter, cDNA and 3′WAPUTR was released from pWAPFIX by EcoR I digestion, subjected to gelelectrophoresis, removed from the gel and purified. This fragment wasligated with Kpn I digested pUC18 and the reaction mixture used totransform E. coli JM109 by electroportation. After electroportation,cells were plated on LB ampicillin plates with picked colonies grown inTB ampicillin broth. Plasmids from picked colonies were purified andsubjected to EcoRI enzyme digestion and electrophoresis. After insertconfirmation, large scale purification was undertaken, according tomethods well known to the skilled artisan.

2. Preparation of Factor IX-Encoding DNA for Microinjection

Chimeric constructs containing either the 7.2 kb mouse WAP gene, orcontaining the WAP promoter, human Factor IX gene and 3′ WAP sequencewere excised from pUCWAPFIX by EcoRI restriction digest and purified formicroinjection using low melting point agarose electrophoresis. The DNA:agarose band was cut from the gel slab. The agarose band was thentreated with agarase to degrade and remove agarose contamination.

After digestion, the solution containing the cDNA was brought to 10 mMMg2+, 20 mM EDTA and 0.1% SDS and then extracted with phenol/chloroform.DNA was precipitated from the aqueous layer with 2.5 volumes of ethanolin the presence of 0.3 M sodium acetate at −20 degrees centigradeovernight. After centrifugation, the pellet was washed with 70% ethanol,dried, and each of the constructs was resuspended and dissolved inBrinsters microinjection buffer to a concentration of 1.4 or 7 μg/ml(for mice), 14 μg/ml (for pigs).

According to another protocol, extracted DNA was purified by HPLC, asfollows. After cleaving a chimeric gene from its vector, the solutionwas brought to 10 mM magnesium, 20 mM EDTA and 0.1% SDS and thenextracted with phenol/chloroform. DNA was precipitated from the aqueouslayer with 2.5 volumes of ethanol in the presence of 0.3 M sodiumacetate at −20° C. overnight. After centrifugation, the pellet waswashed with 70% ethanol, dried, and resuspended in sterile distilledwater.

The digested DNA was precipitated with isopropanol and then dissolved inTE buffer at 0.3 μg/ml. Fragments were purified by HPLC using a WatersGEN FAX PAC HPLC column. The column was run isocratically using a bufferconsisting of 25 mM Tris-HCl (pH 7.5), 1 mM sodium EDTA, and 0.63 MNaCl. About 15 μg of digested DNA was loaded on the column at a time.DNA samples from all of the chromatographic runs were then pooled,reprecipitated, and run through the column a second time.

DNA concentrations were determined by agarose gel electrophoresis bystaining with ethidium bromide and comparing the fluorescent intensityof an aliquot of the DNA with the intensity of standards. Samples werethen adjusted to 10 μg/ml and stored at −20° C., prior tomicroinjection.

Example 2 Production of Transgenic Pigs that Express the Human Factor IXGene

Pig embryos were recovered from the oviduct, and were placed into a 1.5ml microfuge tube containing approximately 0.5 ml embryo transfer media(Beltsville Embryo Culture Medium). Embryos were centrifuged for 12minutes at 16,000×g RCF (13,450 RPM) in a microcentrifuge (Hermle, modelZ231). The embryos were then removed from the microfuge tube with adrawn and polished Pasteur pipette and placed into a 35 mm petri dishfor examination. If the cytoplasm was still opaque with lipid such thatpronuclei were not visible, the embryos were centrifuged again for 15minutes. Embryos were then placed into a microdrop of media(approximately 100 μl) in the center of the lid of a 100 mm petri dish,and silicone oil was used to cover the microdrop and fill the lid toprevent media from evaporating. The petri dish lid containing theembryos was set onto an inverted microscope (Carl Zeiss) equipped withboth a heated stage and Hoffman Modulation Contrast optics (200× finalmagnification). A finely drawn (Kopf Vertical Pipette Puller, model 720)and polished (Narishige microforge, model MF-35) micropipette was usedto stabilize the embryos while about 1-2 picoliters of HPLC-purified DNAsolution containing approximately 200-500 copies of a mixture of the twochimeric constructs was delivered into the male pronucleus with anotherfinely drawn micropipette. Embryos surviving the microinjection processas judged by morphological observation were loaded into a polypropylenetube (2 mm ID) for transfer into the recipient pig.

Example 3 Production of pUCWAP6 “Cassette Vector” and Plasmid pUCWAP6FIX

Production of pUCWAP6 “Cassette Vector”

Generally, the entire murine WAP gene was cloned by standard methods, asdescribed above in Example 1, and regulatory 5= and 3= flankingsequences of the mouse WAP gene were used for mammary specificexpression. Specifically, a cassette vector containing a mouse WAPpromoter, defined as a 4.1 kb NotI-KpnI fragment immediately 5= to theWAP signal sequence and a 1.6 kb fragment of the 3=untranslated regionof the WAP gene was prepared. These regulatory sequences do not includecoding and intragenic untranslated sequences (introns) of the WAP gene.

The vector designated pUCWAP6 was derived from genetic elements from thefollowing plasmids as starting material: pUC18, pWAP4 and p227.6, whichwere provided by the American Red Cross. The development of pUCWAP6 isas follows: The pUC18 vector was cut with the enzymes EcoRI and Hind IIIto remove the multiple cloning site of the vector, blunted withexonuclease and ligated with NotI linkers. The linearized plasmid wasthen cut with NotI and ligated. Ligation mixture was used to transformE. coli DH5α cells on LB ampicillin plates, picked colonies were grownin TB ampicillin broth, plasmids were isolated and cut with NotI thensubjected to gel electrophoresis. Plasmid was judged to be correct anddesignated as pUCNotI (See FIG. 3A). The vector pWAP4 was cut with EcoRIand the fragment containing the WAP 5=2.6 kbp and 3= genetic elementswere separated by gel electrophoresis and purified. The ends of thefragment were modified by blunting with exonuclease and NotI linkerswere ligated on. The fragment was cut with NotI and ligated into theNotI restriction site of pUCNotI then used to transform E. coli DH5αcells on ampicillin plates picked colonies were grown in TB ampicillinbroth. Isolated plasmid was verified to be correct by NotI digestionwith the plasmid being designated pUCWAP5. The pUC WAP5 plasmid wassubjected to KpnI digestion and a partial NotI digestion producing afragment that contained the pUCNotI vector sequence flanked by the mWAP3=UTR (See FIG. 3B). This fragment was ligated with the 4.1 kb 5=WAPpromoter produced from digestion of p227.6 with NotI, KpnI and Hind III.The ligation mixture was then used to transform E. coli JM109 cells thatwere grown on LB ampicillin plates picked colonies were grown in TBampicillin broth, plasmids isolated were cut with Not I, and NotI/KpnIand judged to be correct. The plasmid was then designated pUCWAP6 (SeeFIG. 3C).

Production of pUCWAP6FIX

As shown in FIG. 4, the plasmid pUCWAP6FIX was produced by digestion ofpUCWAPFIX with KpnI and isolating the FIX cDNA by gel electrophoresis.This fragment was inserted into the KpnI site of pUCWAP6 after KpnIdigestion and both fragments were then subjected to ligation. Theligation mixture was then used to transform E. coli JM109 cells thatwere then plated on LB ampicillin plates. Picked colonies were grown inTB ampicillin broth and plasmids were isolated. Isolated plasmids weredigested with NsiI to verify orientation of the cDNA insert. Plasmidsthat contained the insert in the correct orientation were designatedpUCWAP6FIX. After insert confirmation, large scale purification wasundertaken, according to methods well known in the art. DNA was preparedfor microinjection as described above.

Example 4 Production of Transgenic Mice That Express the Human Factor IXGene

Transgenic mice were produced essentially as described by Hogan et al.,Manipulating the Mouse Embryo, Cold Spring Harbor Press, (1986), whichis hereby incorporated by reference. That is, glass needles formicro-injection were prepared using a micropipet puller and microforge.Injections were performed using a Nikon microscope having HoffmanModulation Contrast optics, with Narashigi micromanipulators and apico-injector driven by N2 (Narashigi).

Fertilized mouse embryos were surgically removed from oviducts ofsuperovulated female CD-1 mice and placed into M2 medium. Cumulus cellswere removed from the embryos with hyaluronidase at 300 μg/ml. Theembryos were then rinsed in new M2 medium, and transferred into M15medium for storage at 37 degrees centigrade prior to injection.

Stock solutions containing about 1.4 μg/ml of the above described DNAwere prepared and microinjected into the pronuclei of 1 cell mouseembryos. In addition, stock solutions containing about 7 μg/ml total DNAwere prepared and microinjected into the pronuclei of mouse embryos.

After injecting the DNA solution into the male pronucleus, embryos wereimplanted into avertin-anesthesized CD-1 recipient females madepseudo-pregnant by mating with vasectomized males. About 25-30microinjected mouse embryos per recipient were transferred intopseudopregnant females. Embryos were allowed to come to term and thenewborn mice were analyzed for the presence of the transgene by PCRusing the primers FIXS1 and FIXA1 described in Table 1, below.

Example 5 Preparation of DNA from Transgenic Animals

DNA can be prepared from tissue of a transgenic animal of any species bythe method exemplified below for mice. Marmur., J. Mol. Biol. 3: 208(1961), incorporated herein by reference.

A 5 mm piece of mouse tail was removed from young, potentiallytransgenic mice at weaning (3 weeks) age, and frozen in liquid nitrogen.To the frozen tissue was added 840 μl of Lysing Solution (8 mM EDTA-0.8%2-mercaptoethanol-80 μg/ml Proteinase K-1 M sodium chlorate in 40 mMTRIS buffer) pH 8.0 and 120 mM NaCl, and the mixture incubated at 50degrees centigrade. The mixture was then extracted with 250 μl ofphenol/chloroform-/isoamyl alcohol (25:24:1) for 10-15 seconds, thencentrifuged for 10 minutes. The supernatant fluid (about 830 μl) wasremoved to a fresh tube, and a DNA clot produced by vortexing thesolution with 0.6 vols. of isopropanol. The mother liquor was decanted,and the DNA clot rinsed twice with 80% ethanol. The DNA clot wasisolated by 5 minutes or centrifugation, aspiration of the supernatantfluid, and air drying of the clot with a stream of air for 10 minutes.

The DNA clot was dissolved in 250 μl of the TE buffer (10 mM Tris. HCl,pH 7.0-1 mM EDTA, and the solution treated with 10 μl of RNase (1 mg/mlRNase A and 4,0000 units/ml RNAse T1) for 1 hour at 37 degreescentigrade. This mixture was shaken with 50 μl of a 24:1 (v/v) solutionof chloroform-isoamyl alcohol for 5-10 seconds, centrifuged, and thesupernatant fluid transferred to a fresh tube.

The recovered supernatant fluid above was mixed sequentially with 25 μlof 3M sodium acetate and 0.5 ml of 95% ethanol. The supernatant fluidabove was mixed sequentially with 25 μl of 3M sodium acetate and 0.5 mlof 95% ethanol. The supernatant fluid was decanted from the precipitatedDNA, and the precipitate washed with 80% ethanol. The purified DNA wasisolated by centrifugation, air dried, then dissolved in 150 μl of TE.

Essentially the same technique was used to prepare DNA from pigs, andthe same or similar techniques can be used to prepare DNA from otheranimals. Such DNA can be analyzed to determine whether transgenicanimals carried recombinant structures.

Example 6 Analysis of DNA Derived from Tissue

To determine whether test animals carried the recombinant constructs,tissue samples were removed from transgenic animals and treated withproteinase K and SDS at 37° C. overnight. The mixture was then incubatedwith DNase-free RNase at 37° C. for 1-2 hours. DNA was precipitated fromthe mixture with sodium acetate and ethanol at −20° C. overnight,collected by centrifugation, washed in 70% ethanol and dried. The driedDNA pellet was used directly for polymerase chain reaction (PCR). Insome cases, the mixture was extracted extensively with phenol/chloroformprior to ethanol precipitation.

Oligonucleotide pairs were used to prime polymerase chain reactions thatdetected the presence of the WAP gene or the Factor IX gene in thetransgenic animals. See Table 1, below. Reactions were performed usingan annealing temperature of 58° C., a denaturation temperature of 94°C., and an extension temperature of 72° C., using 100 ng of oligoprimers and 50 ng of (genomic) template DNA per reaction, and cyclingthrough the temperatures 40 times using an automatic temperature cycler(M.J. Research). PCR reactions were analyzed by running 20% of thereaction products on agarose gels and identifying fragment sizes bycomparison with marker DNA fragments.

Two founder transgenic pigs (one male and one female) contained a 2.6 kbmouse WAP promoter-Factor IX cDNA-1.6 kb WAP gene 3-′ end construct thathad been coinjected with the 7.2 kb mouse WAP gene (EcoRI-EcoRI)fragment. As shown in Table 2, the male, 57-7, did not transmit thetransgene. In contrast, founder 58-1 has produced one female offspringhaving the Factor IX cDNA transgene. Founder 58-1 has produced sixadditional offspring, three females and three males, from her secondlitter. The three females were not transgenic. Two of the males from thesecond litter tested positive for the Factor IX transgene.

TABLE 1 Primer Sequences humFIX5′KpnI 5′gcta\ggtacc\atgcagcgcg (SEQ IDNO: 1) humFIX3′KpnI 5′gtca\ggtacc\ttaagtgagct (SEQ ID NO :2) FIXS15′ggataacatcactcaaagcac (SEQ ID NO: 3) WAP3′A15′tagcagcagattgaaagcattatg (SEQ ID NO: 4) FIXA1 5′gtgaactttgtagatc (SEQID NO: 5)

TABLE 2 Transgenic Pigs Containing Recombinant Human Factor IX DNA PigID Construct Sex Comments 57-7 WAP/FIX Male Founder, PCR* positive forWAP and FIX 58-1 WAP/FIX Female Founder, PCR positive for WAP and FIX63-1 WAP/FIX Female G¹ from 58-1, positive for WAP and FIX 63-2 WAP/FIXFemale G¹ from 58-1, positive for WAP and FIX (dead) litter#10 WAP/FIX 3Female, 2 transgenic males to 58-1 3 Male WAP: Whey acid protein; FIX:Factor IX; *Detection of human Factor IX transgene carried out by thePCR method.

Example 7 Expression of Human Factor IX in the Milk of Transgenic Pigs

Daily expression levels of recombinant human Factor IX in the milk oftransgenic pig 58-1 were determined as follows. Lactating sows wereinjected intramuscularly with 30-60 IU of oxytocin (Vedco Inc., St.Joseph, Mo.) to stimulate milk let-down. Letdown occurred two to fiveminutes after injection. Pigs were milked by hand during the course ofthis study. Immediately after collection the milk was diluted 1:1 with200 mM EDTA, pH 7.0 to solubilize the caseins and then frozen. Smallaliquots (about one milliliter) of the milk/EDTA mixture were taken andcentrifuged for approximately 30 minutes at 16000×g at 4° C. The fatlayer was separated from the diluted whey fraction, and the diluted wheyfraction was used for all further assays. In this study, allconcentration values reported for milk were obtained from diluted wheysamples that were multiplied by a factor of 1.9 to account for dilutionwith EDTA and subsequent removal of milk fat.

Amounts of Factor IX in milk were measured by polyclonal ELISA. Briefly,Immulon II microtiter plates (Fisher Scientific, Pittsburgh) were coatedovernight with 100 μl/well of 1:1000 rabbit anti-human Factor IX (Dako)in 0.1 M NaHCO₃, 0.1 M NaCl, pH 9.6 at 4° C. The wells were washed withTBS-Tween (TBST, 25 mM Tris, 50 mM NaCl, 0.2% Tween 20, pH 7.2), andthen blocked for 30 minutes with TBS/0.1% BSA at room temperature.Samples and human Factor IX standard (a gift from the American RedCross) in the TBS-BSA dilution buffer were added in triplicate to thewells (100 L/well) and incubated at 37° C. for 30 minutes. The wellswere then washed and blocked for another 10 minutes at room temperature.Goat anti-human Factor IX (American Diagnostica, Greenwich, Conn.),1:1000 in TBS-BSA, was then incubated in the wells for 30 minutes at 37°C., followed by anti-goat IgG/HRP (Sigma, St. Louis). Bound chromophorewas detected with OPD substrate (Abbott, Chicago) at 490 nm using anEL308 Bio-Tek Microplate reader.

As shown in Table 3, daily expression levels of 100-220 μg/ml milk weremaintained throughout the 10 day lactation.

TABLE 3 Recombinant Factor IX Levels in Milk of Transgenic Pig 58-1,First Lactation rhFIX Day of Level² Lactation [μg/ml] 3 160 ± 26 4 145 ±20 5 100 ± 25 6 135 ± 15 7 220 ± 30 9 170 ± 35 10 185 ± 50 ²Recombinanthuman Factor IX (rhFIX) levels were determined by ELISA on daily samplesof EDTA-diluted whey.

Example 8 Western Analysis of Human Factor IX Produced by TransgenicPigs

Recombinant human Factor IX also was examined using Western analysis.Daily samples of EDTA-diluted whey from 58-1 were electrophoresed on8-16% SDS gels (Novex, San Diego). Approximately 125 ng of recombinanthuman Factor IX (as determined by polyclonal ELISA) and human Factor IXstandard (American Red Cross), were loaded in each lane. A total of 25μg of total protein from a pool of non-transgenic (NTG) whey was loadedon the gels. After electrophoresis, proteins were transferred overnightto PVDF membranes (Bio Rad). The membranes were washed for 30 minutes inTBST, blocked with TBS/0.05% Tween 20/0.5% Casein (TBST-Casein). Themembranes were developed with rabbit anti-Factor IX (Dako) (1:1000 inTBST-Casein for 45 minutes at 37° C.), followed by anti-rabbit IgG/HRP(Sigma) (1:1000 in TBST-Casein for 45 minutes at 37° C.), and the DABmetal enhanced staining (Pierce). Molecular weight markers werepurchased from Bio Rad.

Western analyses revealed the presence of three sub-populations ofrecombinant human Factor IX: the major population migrated at a M_(r) ofabout 60-65 kDa, which is a slightly lower M_(r) than human Factor IX,and minor sub-populations migrated at about 40-45 kDa, and at about 25kDa. Plasma human Factor IX also possessed a subpopulation at about45-50 kDa.

In yet another study, whole milk from transgenic pig 58-1 was diluted1:1 with 200 mM EDTA, pH 7.0 to dissociate casein micelles. Milk wasskimmed of fat by centrifugation at 4000×g for 30 min, at 2° C. 100 μgsof milk protein were loaded per lane of a 4%/10% SDS-PAGE gel andresolved at 15 mA/hr for one hour and 30 mA/hr for 2 hours. Proteinswere transferred onto nitrocellulose paper (Amersham), at 24 V/h, 4° C.and western blotted to detect rFIX in milk, using an HRP-conjugated goatanti-FIX antibody (Affinity Biologicals) at 0.9 μg/ml concentration. Theresults of this study are set forth in FIG. 1, wherein lanes 1-8represent milk from day 3, 4, 5, 6, 7, 9, 10, 11 of lactation; lane 9,purified recombinant FIX, 1.0 μg; and lane 10, human FIX purified fromplasma, 0.5 μg. The positions of broad range molecular weight markers(BioRad) are indicated on the left.

Example 9 Purification of Human Factor IX from Milk of Transgenic Pigs

Recombinant human Factor IX was purified from a pool of the firstlactation from the milk of 58-1 using ion exchange chromatographyfollowed by metal-dependent immunoaffinity chromatography (MAb 1H5). Inthese studies, all columns and buffers were kept at 4° C. A pool ofdaily EDTA-expanded whey samples was diluted to OD 280 nm of 5.0 withTBS, pH 7.2, then loaded at 1 cm/min on DEAE FF Sepharose. The columnwas washed with TBS, pH 7.2, and then eluted with 0.25 M NaCl in TBS.This fraction was diluted 1:1 with 40 mM MgCl₂ in TBS to a finalconcentration of 20 mM MgCl₂ and loaded on a 1H5 MAb column. The columnwas washed with TBS containing 20 mM MgCl₂, and the product was elutedwith 20 mM citrate, 0.15 M NaCl, pH 6.8. The product was dialyzedovernight against 10 mM imidazole, pH 7.2.

The yields from the anion exchange and immunoaffinity steps werequantitative, and no recombinant human Factor IX was detected in theflow-through chromatographic fractions by polyclonal ELISA. Thistwo-step chromatographic procedure isolated the recombinant human FactorIX to about 80-90% purity.

Example 10 The Biological Activity of Purified Recombinant Human FactorIX

The biological activity of the purified recombinant human Factor IX from58-1 was measured using a one-stage activated partial thromboplastinclotting time assay (APTT) clotting assay following a protocol given bythe American Red Cross Plasma Derivatives Laboratory (Procedure forFactor IX Coagulation Assay, March 1992). Briefly, each well of aplastic Coag-a-mate tray received 90 μl of Factor IX-deficient plasmaplus 10 μl of a Factor IX standard or sample, diluted withTris/saline/BSA. The tray was then placed on an automated analyzer (APTTmode, 240 second activation). The run was started, which automaticallyperformed the addition of 100 μl of APTT reagent and 100 μl of 0.025 MCaCl₂. Data obtained using a standard Factor IX preparation were fittedto the equation y−ax+b where y=clotting time and x=Factor IX, which wasthen used to determine the amount of Factor IX in a sample. TheStandards of normal plasma reference pool (Sigma) and human Factor IX(American Red Cross Plasma Derivatives Laboratory) were used in theassay. Duplicates of 58-1 recombinant human Factor IX, human Factor IX,and normal plasma reference pool samples were run at each dilution.

As shown in Table 4, the immunopurified recombinant human Factor IX hada specific activity of 337 U/mg, which is comparable to theimmunopurified human Factor IX from plasma which had a specific activityof 230 U/mg, and the normal plasma reference pool activity of 250 U/mg.

TABLE 4 Specific Activity of Recombinant Human Factor IX Purified fromthe Milk of a Transgenic Pig Slope Activity Specific Sample Slope RatioEquation (%) Activity NPRP 0.094 1.0 y = 0.094x − 3.7 100% 250 U/mg hFIX0.086 0.92 y = 0.086x − 3.6  92% 230 U/mg rhFIX 0.127 1.35 y = 0.127x −3.4 135% 337 U/mg NPRP: normal plasma reference pool hFIX: human FactorIX standard rhFIX: Factor IX isolated from the transgenic pig

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention, which isdefined by the following claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those in the art to which theinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference in its entirety.

1. A non-human transgenic mammal containing an exogenous DNA moleculethat is stably integrated in its genome, wherein said exogenous DNAmolecule comprises: (a) 5′ regulatory sequences of a mammarygland-specific gene including a promoter; (b) a Factor IX-encoding DNAsequence that encodes a signal sequence, a Factor IX pro-sequence, and aFactor IX sequence in a 5′ to 3′ direction, wherein said signal sequenceis effective in directing the secretion of said Factor IX into the milkof said transgenic mammal and wherein said Factor IX sequence lacks atleast a portion of the complete 5′-untranslated and 3′-untranslatedregions of the Factor IX gene; and (c) 3′ regulatory sequences from amammary gland-specific gene or 3′ regulatory sequences active in amammary gland; wherein said 5′ and said 3′ regulatory sequences areoperatively linked to said Factor IX-encoding DNA sequence.
 2. Thenon-human transgenic mammal of claim 1, wherein said promoter isselected from the group consisting of short whey acidic protein (WAP)promoter, long WAP promoter, short α-casein promoter, short β-caseinpromoter, short kappa-casein promoter, long α-casein promoter, longβ-casein promoter, long kappa-casein promoter, α-lactalbumin promoterand β-lactoglobulin promoter.
 3. The non-human transgenic mammal ofclaim 2, wherein said short WAP promoter is the 2.6 kb EcoRI-KpnIpromoter of the mouse WAP gene.
 4. The non-human transgenic mammal ofclaim 2, wherein said long WAP promoter is the 4.1 kb NotI-KpnI promoteror the 4.2 kb Sau3A-KpnI promoter of the mouse WAP gene. 5-10.(canceled)
 11. The non-human transgenic mammal of claim 1, wherein saidtransgenic mammal is selected from the group consisting of mice, rats,rabbits, pigs, sheep, goats and cows.
 12. The non-human transgenicmammal of claim 1, wherein said transgenic mammal secretes from about100 to about 220 μg of active human Factor IX per milliliter milk.13-16. (canceled)
 17. The non-human transgenic mammal of claim 12,wherein said transgenic mammal is a pig. 18-19. (canceled)
 20. Thenon-human transgenic mammal of claim 1, wherein said active human FactorIX purified from the milk of said transgenic mammal has a specificactivity that is at least about 15-50% greater than the specificactivity of human Factor IX isolated from human plasma. 21-22.(canceled)
 23. A process for producing Factor IX, comprising: (a)providing a non-human transgenic mammal having integrated into itsgenome an exogenous DNA molecule, wherein said exogenous DNA moleculecomprises: (1) 5′ regulatory sequences of a mammary gland-specific geneincluding a promoter; (2) a Factor IX-encoding DNA sequence that encodesa signal sequence, a Factor IX pro-sequence and a Factor IX sequence ina 5′ to 3′ direction, wherein said signal sequence is effective indirecting the secretion of said Factor IX into the milk of saidtransgenic mammal and wherein said Factor IX sequence lacks at least aportion of the complete or the complete 5′-untranslated and3′-untranslated regions of the Factor IX gene; and (3) 3′ regulatorysequences from a mammary gland-specific gene or 3′ regulatory sequencesactive in a mammary gland; wherein said 5′ and said 3′ regulatorysequences are operatively linked to said Factor IX-encoding DNAsequence; (b) allowing said DNA sequences encoding said Factor IX to beexpressed and said Factor IX to be secreted into the milk of saidtransgenic mammal; (c) collecting said milk from said mammal; and (d)isolating said Factor IX from said milk.
 24. The process of claim 23,wherein said promoter is selected from the group consisting of shortwhey acidic protein (WAP) promoter, long WAP promoter, short α-caseinpromoter, short β-casein promoter, short kappa-casein promoter, longα-casein promoter, long β-casein promoter, long kappa-casein promoter,α-lactalbumin promoter and β-lactoglobulin promoter.
 25. The process ofclaim 24, wherein said short WAP promoter is the 2.6 kb EcoRI-KpnIpromoter of the mouse WAP gene.
 26. The process of claim 24, whereinsaid long WAP promoter is the 4.1 kb NotI-KpnI promoter or the 4.2 kbSau3A-KpnI promoter of the mouse WAP gene. 27-32. (canceled)
 33. Theprocess of claim 23, wherein said transgenic mammal is selected from thegroup consisting of mice, rats, rabbits, pigs, sheep, goats and cows.34. The process of claim 23, wherein said transgenic mammal secretesfrom about 100 to about 220 μg of active human Factor IX per millilitermilk. 35-44. (canceled)
 45. A method of treating a patient havinghemophilia B comprising administering to said patients a hemophilia Bsymptom preventing or ameliorating amount of Factor IX produced by thetransgenic non-human mammal of claim 1 and a pharmaceutically acceptablecarrier.
 46. Mammary gland cells obtained from the non-human transgenicmammal of claim 1, wherein said cells produce said Factor IX. 47-49.(canceled)
 50. A biologically active human Factor IX produced in thenon-human transgenic mammal of claim 1, said Factor IX comprising aspecific activity that is at least about 5-200% greater than thespecific activity of human Factor IX isolated from human plasma.
 51. Abiologically active human Factor IX of claim 50, wherein said non-humantransgenic mammal is selected from the group consisting of mice, rats,rabbits, pigs, sheep, goats and cows.